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Chromatin Structure

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Chromatin Structure & Dynamics Victor Jin Department of Biomedical Informatics The Ohio State University * * DNA methylation transfer of a methyl group to the carbon ... – PowerPoint PPT presentation

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Title: Chromatin Structure


1
Chromatin Structure Dynamics
Victor Jin Department of Biomedical
Informatics The Ohio State University
2
Chromatin
  • Walther Flemming first used the term Chromatin in
    1882. At that time, Flemming assumed that within
    the nucleus there was some kind of a
    nuclear-scaffold.
  • Chromatin is the complex of DNA and protein that
    makes up chromosomes.
  • Chromatin structure DNA wrapping around
    nucleosomes a beads on a string structure.
  • In non-dividing cells there are two types of
    chromatin euchromatin and heterochromatin.

3
Chromatin Fibers
Chromatin as seen in the electron microscope.
(source Alberts et al., Molecular Biology of
The Cell, 3rd Edition)
11 nm (beads)
30 nm chromatin fiber
4
Nucleosome
  • The basic repeating unit of chromatin.
  • It is made up by five histone proteins H2A, H2B,
    H3, H4 as core histones and H1 as a linker.
  • It provides the lowest level of compaction of
    double-strand DNA into the cell nucleus.
  • It often associates with transcription.

H3
H2A
H2B
H4
1974 Roger Kornberg discovers nucleosome who won
Nobel Prize in 2006.
5
Core Histones are highly conserved proteins -
share a structural motif called a histone fold
including three a helices connected by two loops
and an N-terminal tail
6
Histone Octamer
lt 11 nm
gt
  • Each core histone forms pairs as a dimer
  • contains 3 regions of interaction with dsDNA
  • H3 and H4 further assemble tetramers.
  • The histone octamer
  • organizes 146 bp of DNA
  • in 1.65 helical turn of DNA
  • 48 nm of DNA packaged in a disc of 6 x 11nm

lt 6 nm gt
7
Nucleosome Assembly In Vitro
4 core histones 1 naked DNA template at 4C at
2M salt concentration, from Dyer et al, Methods
in Enzymology (2004), 37523-44.
8
DNA compaction in a human cell nucleus
10,000 nm
11 nm
30nm
1bp (0.3nm)
9
The N-terminal tails protrude from the core
10
Histone Modifications
Acetylation
Ac
Me
Methylation
Me
Histone Code
Ub
Ubiquitination

Su
Sumoylation
P
Phosphorylation
11
Acetylation of Lysines
  • Acetylation of the lysines at the N terminus of
    histones removes positive charges, thereby
    reducing the affinity between histones and DNA.
  • This makes RNA polymerase and transcription
    factors easier to access the promoter region. 
  • Histone acetylation enhances transcription while
    histone deacetylation represses transcription.

12
Methylation of Arginines and Lysines
  • Arginine can be methylated to form mono-methyl,
    symmetrical di-methyl and asymmetrical
    di-methylarginine.
  • Lysine can be methylated to form mono-methyl,
  • di-methyl
  • and tri-methylarginine.

13
Methylation of Histone H3-K27
EZH2
K27
14
Functional Consequences of Histone Modification
  • Establishing global chromatin environment, such
    as Euchromatin, Heterochromatin and Bivalent
    domains in embryonic stem cells (ESCs).
  • Orchestration of DNA-based process transcription.

15
Euchromatin
  • A lightly packed form of chromatin
  • Gene-rich
  • At chromosome arms
  • Associated with active transcription.

16
Heterochromatin
  • A tightly packed form of chromatin
  • At centromeres and telomeres
  • Contains repetitious sequences
  • Gene-poor
  • Associated with repressed transcription.

17
Bivalent Domains
Poised state. The chromatin of embryonic stem
cells has bivalent domains with marks of both
gene activation and repression. In these domains,
the tail of histone protein H3 has a methyl group
attached to lysine 4 (K4) that is activating and
a methyl group at lysine 27 (K27) that is
repressive (above). This contradictory state may
keep the genes silenced but poised to activate if
needed. When the cell differentiates (right),
only one tag or the other remains, depending on
whether the gene is expressed or not.
18
DNA Methylation
DNA methyltransferase
S-adenosylmethionine
5-methylcytosine
deoxycytosine
19
CpG Islands
  • CpG island a cluster of CpG residues often
    found near gene promoters (at least 200 bp and
    with a GC percentage that is greater than 50 and
    with an observed/expected CpG ratio that is
    greater than 0.6).
  • 29,000 CpG islands in human genome (60 of
    all genes are associated with CpG islands)
  • Most CpG islands are unmethylated in normal
    cells.

20
Chromatin modifications
Mark Transcriptionally relevant sites Biological Role
Methylated cytosine (meC) CpG islands Transcriptional Repression
Acetylated lysine (Kac) H3 (9,14,18,56), H4 (5,8,13,16), H2A, H2B Transcriptional Activation
Phosphorylated serine/threonine (S/Tph) H3 (3,10,28), H2A, H2B Transcriptional Activation
Methylated argine (Rme) H3 (17,23), H4 (3) Transcriptional Activation
Methylated lysine (Kme) H3 (4,36,79) H3 (9,27), H4 (20) Transcriptional Activation Transcriptional Repression
Ubiquitylated lysine (Kub) H2B (123/120) H2A (119) Transcriptional Activation Transcriptional Repression
Sumoylated lysine (Ksu) H2B (6/7), H2A (126) Transcriptional Repression
21
Genome-wide Distribution Pattern of Histone
Modification Associated with Transcription
Source Li et al. Cell (Review, 2007), 128707-719
Li et al. Cell (review) 128, 707-719
22
Epigenetics
  • Modifications of DNA (cytosine methylation) and
    proteins (histones) define the epigenetic
    profile.
  • In 1942, Conrad Waddington first used
    epigenetics to describe the interactions
    between genome and environment that give rise to
    differences between cells during embryonic
    development.
  • Currently, Epigenetics is the study of heritable
    changes in gene function that occur without a
    change in DNA sequence.
  • Summarizes mechanisms and phenomena that affect
    the phenotype of a cell or an organism without
    affecting the genotype.
  • Epigenomics is the study of these epigenetic
    changes on a genome-wide scale.

23
Normal Cellular Functions Regulated by Epigenetic
Mechanisms
  • Correct organization of chromatin
  • Genomic imprinting
  • Silencing of repetitive elements
  • X chromosome inactivation

24
X-chromosome Inactivation
  • X-inactivation (also called lyonization) is a
    process by which one of the two copies of the X
    chromosome present in female mammals is
    inactivated.
  • The inactive X chromosome is silenced by
    packaging in repressive heterochromatin.
  • The choice of which X chromosome will be
    inactivated is random in higher mammals such as
    mice and humans. Once an X chromosome is
    inactivated it will remain inactive throughout
    the lifetime of the cell.
  • Silencing initiated at Xic/XIC and spreads along
    chromososme.
  • 5meC CpG DNA modification is observed in
    inactivated X chromosomes.
  • 5meC binds transcriptional repressor MeCP2
    (MethylC-binding Protein-2).
  • MeCP2 binds Sin3 with RPD3 histone deacetylase.

Source Jones et al. Nat.Genet. 19, 187 (1998)
25
Epigenetic Diseases
  • Some human disorders such as Angelman syndrome
    and Prader-Willi syndrom are associated with
    genomic imprinting.
  • Involvement in cancer and development
    abnormalities.
  • The emerging hypothesis of cancer stem cells
    (CSC).

26
DNA Methylation and Gene Silencing in Cancer
Cells
CpG island
CG
CG
CG
CG
MCG
MCG
CG
Normal
1
3
2
4
MCG
MCG
MCG
MCG
CG
CG
CG
Cancer
1
2
3
4
X
C cytosine mC methylcytosine
27
Progressive Alterations in DNA Methylation in
Cancer
Region-Specific Hypermethylation
Global Hypomethylation

Normal
Cancer
28
Epigenetic Mediation of Gene Silencing
29
CpG Island Methylation A Stable, Heritable and
Positively Detectable Signal
1
2
3
4
Carcinoma
5
Normal Epithelia
Dysplasia
Carcinoma in situ
Metastasis
30
CpG Island Methylation A Stable, Heritable and
Positively Detectable Signal
1
2
3
4
Carcinoma
5
Normal Epithelia
Dysplasia
Carcinoma in situ
Metastasis
31
CpG Island Methylation A Stable, Heritable and
Positively Detectable Signal
1
2
3
4
Carcinoma
5
Normal Epithelia
Dysplasia
Carcinoma in situ
Metastasis
32
CpG Island Methylation A Stable, Heritable and
Positively Detectable Signal
1
2
3
4
Carcinoma
5
Normal Epithelia
Dysplasia
Carcinoma in situ
Metastasis
33
Epigenetic Alterations in Cancer Stem Cells
  • Cancer Stem Cells Stem cells arising through the
    malignant transformation of adult stem cells.
  • Cancer Stem Cells Hypothesis Cancer stem cells
    are the main driving force behind tumor
    proliferation and progression.

34
Hallmarks of Cancer Stem Cells
  • A cell residing in a tumor that
  • has a capacity to remain in an undifferentiated
    state
  • has properties of asymmetric divisions and
    self-renewal
  • has metastatic and repopulation capacities at
    specific niches (microenvironment) in the body
  • gives rise to a tumor that is histologically
    identical to the one from which the CSC is derived

35
The Evidence of Cancer Stem Cells
  • First isolated from the patients of acute myeloid
    leukemia in 1997 by John Dick and colleagues at
    the University of Toronto.
  • Isolated from two solid tumors, breast and brain
    cancers.
  • 1 cancer cells may be really cancer stem cells.

36
More ChIP-chip
  • Step 1 Rapid fixation of cells chemically
    cross-links DNA binding proteins to their genomic
    targets in vivo.
  • Step 2 Cell lysis releases the DNA-protein
    complexes, and sonication fragments the DNA.
  • Step 3 Immunoprecipitation (IP) purifies the
    protein-DNA fragments, with specificity dictated
    by antibody choice.
  • Step 4 Hydrolysis reverses the cross-links
    within the released DNA fragments.
  • Step 5 PCR amplification of ChIP DNA
  • Step 6 PCR amplification on a known
    binding-site region for that protein will need to
    be performed using either conventional PCR
    methods followed by agarose gel electrophoresis
    or by quantitative PCR.
  • Step 7 Labeling pool of protein-DNA fragments.
  • Step 8 Hybridization of DNA onto microarrays
    featuring 60-mer oligonucleotide probes.

37
Major types of array platforms
  • NimbleGen Arrays tiling arrays, promoter
    arrays, whole genome arrays. (http//www.nimblegen
    .com/products/chip/index.html)
  • Agilent Arrays promoter arrays, whole genome
    arrays.
  • (http//www.chem.agilent.com/Scripts/Phome.asp)
  • Affymetrix Arrays tiling arrays, Chr21,22
    arrays, whole genome arrays.
  • (http//www.affymetrix.com/index.affx)

38
Measurement of intensity of probes on the array
  • The hybridized arrays were scanned on an Axon
    GenePix 4000B scanner (Axon Instruments Inc.) at
    wavelengths of 532 nm for control (Cy3), and 635
    nm (Cy5) for each experimental sample.
  • Data points were extracted from the scanned
    images using the NimbleScan 2.0 program
    (NimbleGen Systems, Inc.).
  • Each pair of N probe signals was normalized by
    converting into a scaled log ratio using the
    following formula
  • Si Log2 (Cy5l(i) /Cy3(i))

39
Antibody Validation
  • Confirming on a known target
  • Different antibodies to same factor
  • Antibodies to different family members
  • siRNA-ChIP
  • Antibodies to two components of a complex
  • Antibodies to an enzyme/modification pair

40
Confirming on a known target
41
Comparison of biological replicates and
antibodies to different E2Fs
42
Loss of E2F6 ChIP signal after knockdown of E2F6
siRNA
43
Reproducibility of promoter arrays using
biological replicates
  • H3me3K27 Ntera2 cells
  • Top 1000 overlap
  • Top 1000 overlap
  • Promoter 1
  • Promoter 2

44
Biological reproducibility on tiling arrays
  • 500 kb region of chromosome 6
  • 500 kb region of chromosome 1

45
Amount of Sample Per ChIP
Number of cells Chromatin input ChIP output
1x107 200 µg 150 ng
1x106 20 µg 10 ng
5x105 10 µg 1.3 ng
1x105 2 µg 300 pg
1x104 200 ng 30 pg
46
Amount of Sample Per ChIP
Number of cells Chromatin input ChIP output
1x107 200 µg 150 ng
1x106 20 µg 10 ng
5x105 10 µg 1.3 ng
1x105 2 µg 300 pg
1x104 200 ng 30 pg
47
Miniaturization
  • Standard ChIP Protocol (1x107 cells WGA2)
  • Promoter Arrays
  • Genome Tiling Arrays
  • MicroChIP Protocol (10,000-100,000 cells WGA4)
  • Promoter Arrays
  • Genome Tiling Arrays

48
Reproducibility of MicroChIP Protocol
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